Depression and related disorders are a major public health problem, compounded by the fact that at least half of patients are not effectively treated by currently available medications. Among the most commonly prescribed is the class of selective serotonin (5-HT) reuptake inhibitors (SSRIs), which act to inhibit 5-HT transporter (SERT) mediated 5-HT uptake. The increase in extracellular 5-HT that follows is thought to be critical for initiation of the cascade of downstream events needed for therapeutic effects. Although SERT is the major player regulating high-affinity 5-HT uptake, there is emerging evidence for an important role of organic cation transporter-3 (OCT3) and possibly the plasma membrane monoamine transporter (PMAT) in taking up 5-HT in brain. This raises the possibility that lack of therapeutic response following SERT blockade could be due to significant 5-HT uptake by OCT3 (and/or PMAT). Our studies using decynium-22 (D-22), a blocker of both OCT3 and PMAT, lend support to this idea. For example, D-22 augments the effect of an SSRI, fluvoxamine, to inhibit 5-HT uptake and to produce antidepressant-like effects in wildtype mice. Moreover, D-22 produces these effects also when given alone in mice that lack, or have reduced SERT expression. Thus, the antidepressant-like effect of D-22 appears to be most pronounced when SERT is either pharmacologically or genetically inactivated. We also found that OCT3 expression (but not PMAT) is increased in mice with a constitutive reduction of SERT, suggesting a compensatory role for OCT3. One important aspect of the proposed studies will be to examine the possibility that this also occurs after chronic treatment with SSRIs, which is known to reduce SERT expression. In addition to 5-HT, OCT3 (and PMAT) can transport norepinephrine (NE) and dopamine (DA), neurotransmitters also linked to the therapeutic action of current antidepressants. Taken together, the goals of the proposed studies are to (1) validate OCT3 (and/or PMAT) as the site where D-22 produces its antidepressant-like effect; (2) determine the relative importance of inhibition of 5-HT, NE and DA uptake in producing the antidepressant-like effect of D-22, and (3) examine the therapeutic potential of D-22 by studying its effect on biogenic amine uptake and antidepressant-like activity after its chronic administration. The results of these studies will help to establish OCT3 (and/or PMAT) as a novel target for the discovery of drugs with improved therapeutic potential, as well as provide a mechanism that can, at least in part, account for poor therapeutic response to current antidepressant drugs.